Pathophysiologie des infections fœto-placentaires: l’exemple de la listériose

CEMI 18 – 15 mars 2013 Institut Pasteur

Olivier Disson, PhD Biology of Infection Unit

Institut Pasteur, Inserm U1117

Intestinal barrier asymptomatic

mild gastroenteritis

Blood-brain barrier meningitis, encephalitis

Placental barrier fetal and neonatal infections

Successive steps of human listeriosis

Lymph node

Contaminated food Liver

Spleen

Blood

Blood

Pregnancy-related listeriosis in France

• Correlation of decrease in pregnancy associated cases with dietary recommendations

• In 2012: 38 pregnancy associated cases / lethality around 30% (338 total listeriosis cases)

• A prospective study on risk factors (MonaLisa) is ongoing: 100 pregnancy-associated cases included

Contrôle des produits fromagers

Contrôle des produits carnés

Recommandations auprès des femmes enceintes

Adapted from Goulet et al, EID 2001 Goulet et al, BEH 2012

Listeria monocytogenes

InlA

Human

E-cad

Listeria crossing of the host barriers

• InlA induces internalization in cultured epithelial cells

Listeria monocytogenes

InlB

Human

Met

• InlA induces internalization in cultured epithelial cells

• InlB induces internalization in cultured epithelial cells

Listeria crossing of the host barriers

Listeria monocytogenes

InlA InlB

E-cad

Guinea pig Rabbit

Mouse Rat

Human

E-cad Met Met Met E-cad

Listeria crossing of the host barriers

• InlA and InlB induce internalization in cultured epithelial cells

• InlA-Ecad and InlB-Met interactions are species-specific

Listeria monocytogenes

InlA InlB

E-cad E-cad Met Met Met E-cad

hEcad

Guinea pig Rabbit

Mouse Rat

Human

Lecuit et al, Science 2001

Listeria crossing of the intestinal barrier

• InlA and InlB induce internalization in cultured epithelial cells

• InlA-Ecad and InlB-Met interactions are species-specific

• Critical role for InlA in Listeria crossing of the intestinal barrier, no role for InlB

- Demonstrated in guinea pigs and in humanized mice expressing human E-cadherin only at the intestine level

Unexpected observation

LRRs IR

p120ctn

Actin

E-cadherin

entry

L. monocytogenes

full-length InlA

Some L. monocytogenes isolates express a truncated InlA

LRRs IR

p120ctn

Actin

E-cadherin

truncated InlA

entry

L. monocytogenes

Some L. monocytogenes isolates express a truncated InlA

1- Questions the role of InlA in humans 2- Offers the opportunity to evaluate its role “epidemiologically”

Some L. monocytogenes isolates express a truncated InlA

Clinical strains (N=300)

96% FL InlA

Food strains (N=150)

65% FL InlA

P<1x10-7

• A role for InlA in crossing the intestinal barrier in humans

• Demonstrates the relevance of the iFABP-hEcad Tg model

Bacteremia 93.2% FL InlA

Epidemiological approach

- WB with anti-InlA Mabs Full length (FL) or truncated InlA? - Compare InlA expression profile of strains of clinical or food origin

Bacteremia 93.2% FL InlA

CNS infection 98.2% FL Internalin

Fetoplacental infection 100% FL InlA

• In favor of a role for InlA in L. monocytogenes placental tropism

P<1x10-7 P=4x10-2

Food strains (N=150)

65% FL InlA

Epidemiological approach

- WB with anti-InlA Mabs Full length (FL) or truncated InlA? - Compare InlA expression profile of strains of clinical or food origin

Jacquet et al, JID 2004

The human maternofetal barrier

Histopathological study of placenta from women with listeriosis

Free bacteria Adhesion

Invasion Crossing Multiplication

Invasion Crossing Multiplication

Intravillous abscesses

Amnion infection from the amniotic fluid

Amniotic fluid

Uterine wall

Suggests that L. monocytogenes crosses the materno-fetal barrier at the villous level

If internalin plays a role in targeting the placental barrier, E-cadherin is expected to be expressed at the villous level

accessible hEcad

total hEcad

E-cadherin is expressed at the interface between the maternal blood and the fetal tissue

i.e. the syncytiotrophoblast apical membrane

fetal side maternal side villous explant

Use of human placental explants

Histology

3h: Adhesion to and invasion of the syncytiotrophoblast

Gram staining

IF staining

Histology

24h: Crossing of the placental barrier Development of villous abscesses

similar to actual placental villous

abscesses from women

with listeriosis

Lecuit et al. PNAS 2004

Internalin-dependent infection of the fetal side of the amnion

hEcad labeling of the amnion

fetal side

maternal side

Role of internalin at the human maternofetal interface

Secondary dissemination to the amnion

Targeting and crossing of the placental barrier

An animal model for human fetoplacental listeriosis ?

Double species specificity of InlA and InlB pathways

Ex vivo: InlA+InlB Epidemiological data suggest a role for InlA

In apparent contradiction with human ex vivo and epidemiological data

NO InlA-dependent placental invasion Bakardjiev, IAI 2004

NO InlB-dependent placental invasion Le Monnier, IAI 2004

Hypothesis :

Both InlA AND InlB pathways have to be functional to mediate L. monocytogenes invasion of the human placenta

Need for an animal model permissive to BOTH InlA and InlB Existing Tg mice express hEcad only at the intestinal level…

Use of gerbils, permissive for both InlA and InlB

Listeria crossing of the placental barrier

Real time in vivo imaging of maternofetal listeriosis

DinlAB

Day 1

Day 4

Day 5

WT DAB DAB

Intravenous infection. Bioluminescent bacteria.

WT

WT

Day 1

Day 4

Day 5

DAB DAB

WT WT

WT

DinlAB

Intravenous infection. Bioluminescent bacteria.

Real time in vivo imaging of maternofetal listeriosis

Day 1

Day 4

Day 5

InlA and/or InlB are required for placenta and fetus infection, in contrast to what observed in mice and guinea pig

WT WT

WT

DinlAB

DinlAB

Intravenous infection. Bioluminescent bacteria.

Real time in vivo imaging of maternofetal listeriosis

Listeria crossing of the placental barrier

Placenta 72h post infection

Epithelium Endothelium Listeria Nuclei

• L. monocytogenes can cross the placental barrier in intravenously infected gerbils

Placenta 24h post infection

Epithelium Endothelium Listeria Nuclei

Epithelium Endothelium Listeria

Placenta 42h post infection

Placenta

One point = one organ * P<0,05 ** P<0,01

*** P<0,001

L. monocytogenes invasion of gerbil placenta and fetuses C

I = R

atio

EG

D w

tr / S

trai

ns

Placenta

Rat

io o

f C

mrr a

nd

C

ms

bac

teri

a

One point = one organ * P<0,05 ** P<0,01

*** P<0,001

Fetus

InlA and InlB allow the crossing of the placental barrier

CI =

Rat

io E

GD

wtr /

Str

ain

s L. monocytogenes invasion of gerbil placenta and fetuses

InlB non functional NO role for InlA in placental invasion

in vivo

InlA non functional NO role for InlB in placental invasion

in vivo

InlA AND InlB functional Role for BOTH InlA and InlB in

placental invasion in vivo

and gerbil

and gerbil

Knock-in mice expressing a humanized E16P mEcad

Humanized E16P mouse

E16P mEcad

Genetic approach

- Knock-in strategy homologous recombination in ES cells

- E16P mEcad expression pattern similar to mEcad physiological expression

notably at the intestinal, placental, and blood-brain barrier levels

- Functional receptors for InlA and InlB assess the role of InlA and InlB in a fully permissive animal model

KI mice expressing a humanized E16P mEcad

Placental barrier

Intestinal barrier

Blood-brain barrier

F1 chimera

KI mice expressing a humanized E16P mEcad

Primary enterocytes from humanized E16P mice are permissive to InlA

Listeria innocua L. innocua (InlA)

WT mice

E16P+/+ mice

Ecad Listeria Nuclei

Quantitative assessment of the roles of InlA and InlB at the fetoplacental level

Conclusions & Perspectives

- Fetoplacental invasion is controlled by two species specific interactions, which are indeed interdependent

InlA - E-cadherin InlB - Met

- Fetoplacental invasion is controlled by two species specific interactions, which are indeed interdependent

InlA - E-cadherin InlB - Met

- Specific microbial targeting to the placenta, in addition to “pregnancy-associated immunosuppression”

Conclusions & Perspectives

- Fetoplacental invasion is controlled by two species specific interactions, which are indeed interdependent

InlA - E-cadherin InlB - Met

- Specific microbial targeting to the placenta, in addition to “pregnancy-associated immunosuppression”

- Both pathways have to be functional for fetoplacental invasion, in contrast to what observed for intestinal invasion

Conclusions & Perspectives

Intestinal barrier

Placental barrier

Intestinal barrier

Placental barrier

InlA + InlB -

InlA + InlB +

• Which mechanism(s) explain the difference between intestinal and placental barriers ?

Collaborations

Institut Pasteur Pascale Cossart Charles Babinet Francina Langa-Vives

Biology of Infection Unit, Institut Pasteur, Inserm U1117

Marc Lecuit Solène Grayo Eugénie Huillet Olivier Disson Laetitia Travier Camille Blériot Cindy Fèvre Yu-Huan Tsai Grégoire Gessain Thérèse Couderc Nicolas Gangneux Delphine Judith WHO & French National Reference Centre on Listeria Alexandre Leclercq Caroline Charlier-Woerther Viviane Chenal-Francisque

Funding

Acknowledgments